EP3894539A1 - Specific electroporation and lysis of eukaryotic cells - Google Patents
Specific electroporation and lysis of eukaryotic cellsInfo
- Publication number
- EP3894539A1 EP3894539A1 EP19831604.4A EP19831604A EP3894539A1 EP 3894539 A1 EP3894539 A1 EP 3894539A1 EP 19831604 A EP19831604 A EP 19831604A EP 3894539 A1 EP3894539 A1 EP 3894539A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- lysis
- electric field
- cells
- electrodes
- electroporation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
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- SYOKIDBDQMKNDQ-XWTIBIIYSA-N vildagliptin Chemical compound C1C(O)(C2)CC(C3)CC1CC32NCC(=O)N1CCC[C@H]1C#N SYOKIDBDQMKNDQ-XWTIBIIYSA-N 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/06—Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material
Definitions
- the present invention relates to the field of electro poration and lysis of cells.
- Electroporation also known as electropermeabilization, is commonly used for introducing compounds, such as chemicals, drugs or DNA, into biological cells.
- compounds such as chemicals, drugs or DNA
- cells of a biological sample are exposed to an electrical field which is generated by electrodes that are disposed opposite to each oth er. If the electrical field strength of the applied electrical field is high enough, the membranes of the cells become porous and allow the compounds to cross the membranes of the cells. If the compound is a foreign DNA and the cell is eukaryotic, this process is also widely known as transfection.
- electroporation can either be a reversible or an irreversible process. If the electrical field strength is kept below a cer tain threshold, most of the cells are still alive after exposure to the electrical field, which means that the electroporation is reversible. On the other hand, if the electrical field strength exceeds a certain value, the electroporation process leads to the destruction of the cells and thus becomes irreversible. By means of irreversible electroporation the inner elements of the cells can be released. This process is also known as lysis.
- elec troporation In comparison to viral and chemical approaches for permea- bilization and thermal or chemical approaches for lysis, elec troporation has several advantages, such as superior control over relevant variables, high transfection rate and the absence of contaminants.
- lysis and electroporation of cells in bio logical samples are known. From WO 2015/044191 A1 it is known to perform lysis to non-pathogenic cells in a suspension of cells for further examination of pathogenic cells.
- the non-pathogenic cells are eukaryotic host cells while the pathogenic cells are foreign cells, such as bacteria, a fungus or protozoa.
- the teaching of WO 2015/044191 A1 does not allow to distinguish between cells of the same host material, in particular between eukaryotic cellular bodies of the same host material .
- WO 2007/056027 A1 it is known from WO 2007/056027 A1 to use electrical fields instead of applying heat to liquids for the reduction of cells.
- the electrodes used in WO 2007/056027 A1 are made of a rather thick dielectric material, which is covered with electrically conductive material for connection to a power supply .
- a disadvantage of the assembly in WO 2007/056027 A1 is the requirement of relatively high potential differences between the electrodes due to the large distance between the electrodes.
- the large distance between the electrodes also causes inhomogenei ties in the applied electric fields, which leads to irregular lysis along the electrodes.
- the dielectric materi al makes up the main part of the electrode, as the dielectric material has to be rather thick in order to be coated with elec trically conductive material.
- microfluidic applications such as a tip for a pipette, cannot be realized with the teach ing of WO 2007/056027 A1.
- Another disadvantage of the known methods is that larger cells are more easily lysed than smaller cells due to their size. Therefore, if one desires to lyse small cells in a biolog ical sample made up of small and large cells, the larger cells are inevitably lysed as well.
- the invention provides a method of targeted electroporation and/or lysis of eukaryotic cellular bodies in a biological sam ple with at least two subgroups of eukaryotic cellular bodies, wherein each subgroup has a different susceptibility to electro poration and/or lysis in electric fields, comprising the follow ing steps:
- the biological sample to an electric field in the chamber, wherein the electric field is generated by at least two electrodes which are coated with a dielectric material with a relative permittivity greater than 3.9, preferably greater than 9, more preferably 60 or more and
- the biological sample to an electric field in the chamber, wherein the electric field is generated by at least two electrodes which are coated with a dielectric material with a relative permittivity greater than 3.9, preferably greater than 9, more preferably 60 or more, and
- the electric parameters of the electric field such as the field strength, the frequency or the wave form so that eu karyotic cellular bodies in a biological sample are affected by said electric field for electroporation and/or lysis.
- the invention provides a method of targeted electroporation and/or lysis of eukaryotic cellular bodies in a biological sample with at least a first and a second group or subgroup of eukaryotic cellular bodies, each group or subgroup having an electroporation and/or a lysis rate, wherein the bio logical sample is transferred into a chamber and exposed to an electric field in the chamber which is generated by at least two electrodes which are coated with a dielectric material with a relative permittivity greater than 3.9, preferably greater than 9, more preferably 60 or more, wherein for isolating and/or de livering a compound into the cells of the first group or sub group the electric parameters such as the field strength, the frequency or the wave form of the electric field are chosen to conduct electroporation and/or lysis the first group or subgroup with a different electroporation and/or a lysis rate than the second group or subgroup.
- the invention provides a device, prefera bly handheld device, suitable for targeted electroporation and/or lysis of eukaryotic cellular bodies in a biological sam ple with at least subgroups of eukaryotic cellular bodies, wherein cellular bodies of each subgroup have a different sus ceptibility to electroporation and/or lysis in electrical fields, comprising at least one chamber for receiving the bio logical sample and at least two electrodes for generating an electric field in the chamber, which electrodes are coated with a dielectric material with a permittivity greater than 3.9, preferably greater than 9, more preferably 60 or more, wherein the distance between the electrodes is below 1 mm, preferably below 550 pm, more preferably below 100 pm, wherein the device further comprises an adjustment unit that enables the adjustment of electric parameters of the electric field such as the field strength, the frequency or the wave form so that the subgroups are differently affected with electroporation and/or lysis.
- the device further comprises an adjustment unit that enables the adjustment of electric parameters of
- the invention provides a device, preferably handheld device, for targeted electroporation and/or lysis of cellular bodies that comprises a tip for a pipette with at least one chamber for receiving a biological sample and at least two electrodes for generating an electric field in the chamber, which electrodes form an inner surface of the chamber and which electrodes are coated with a dielectric material with a relative permittivity greater than 3.9, preferably greater than 9, more preferably 60 or more, wherein the distance between the elec- trodes is below 1 mm, preferably below 550 pm, more preferably below 100 pm, wherein the device further comprises an adjustment unit that enables the adjustment of electric parameters of the electric field such as the field strength, the frequency or the wave form.
- the invention provides a device that is an air displacement pipette with a suction and pressure means for creating a reduced pressure in a receiving chamber, wherein receiving chamber is suitable for receiving a biological sample, wherein the chamber has at least two electrodes for generating an electric field in the chamber, which electrodes are each on an inner surface of the chamber and which electrodes are coated with a dielectric material with a relative permittivity greater than 3.9, preferably greater than 9, more preferably 60 or more, wherein the distance between the electrodes is below 1 mm, pref erably below 550 pm, more preferably below 100 pm.
- the device further is connected to or comprises an adjustment unit that enables the adjustment of electric parameters of the electric field such as electric field strength, electric pulse frequency or electric pulse wave form of the electric field.
- the invention provides a method of target ed electroporation and/or lysis of cellular bodies in a biologi cal sample, comprising the following steps:
- the electric field is generated by at least two electrodes which are coated with a dielectric material with a relative permittivity greater than 3.9, preferably greater than 9, more preferably 60 or more, wherein the dis tance between the electrodes is below 1 mm, preferably below 550 pm, more preferably below 100 pm, and
- the electric parameters of the electric field such as the field strength, the frequency or the wave form so that the cellular bodies are lysed and/or electroporated.
- the invention provides a method for manu facturing a device, preferably handheld device, particularly a tip for a pipette, for targeted electroporation and/or lysis of eukaryotic cellular bodies, wherein at least two electrodes are coated with a layer of dielectric material with a permittivity greater than 3.9, preferably greater than 9, more preferably 60 or more, wherein the electrodes are arranged such that the dis tance between the electrodes is below 1 mm, preferably below 550 pm, more preferably below 100 pm.
- eukar yotic cellular bodies comprise nucleated eukaryotic cells, such as lymphocytes, as well as non-nucleated bodies stemming from eukaryotic cells, such as erythrocytes.
- Alternative terms for eukaryotic cellular bodies are eukaryotic cellular vessels or eukaryotic cellular corpuscles.
- the eukaryotic cellular bodies may stem from the same organism, e.g. mammals, humans or ani mals.
- the various types of eukaryotic cellular bodies have in common that they comprise an eukaryotic cell membrane that en closes an intracellular cavity, usually filled with an aqueous medium. The cell membrane is usually from an eukaryotic cell.
- eukaryotic cellular bodies is mostly associated with eukaryotic cells herein, but also includes bodies without a nu cleus, such as erythrocytes. Without being limited to a particu lar theory, it appears that the eukaryotic cell wall is specifi cally susceptible for manipulation by the inventive methods for electroporation or lysis, i.e. inducing leakiness or rupture of the cell membrane, respectively.
- eukaryotic cells and their non-nucleated cellular bodies each have particular discernible susceptibili ties to the electric parameters in a chamber with coated elec trodes according to the invention so that different eukaryotic cellular bodies can be individually targeted without affecting other eukaryotic cellular bodies.
- This allows specific treat- merits and manipulation in a selected type of cells (subgroups in the sample) .
- the size of eu karyotic cellular bodies is not a relevant factor. In prior art, only the larger cells in a mixture could be targeted.
- Cells that can be distinguished are for example tumor cells and non tumor cells (each forming a different subgroup) .
- cells within these group can be distinguished, such as cells from different tissue origins, such as leukocytes, liver cells, kidney cells, erythrocytes, neuronal cells, fat cells, bone cells, cartilage cells, skin cells, epi thelial cells, muscular cells etc..
- the organism can be distinguished, such as animals, like vertebrate, and non
- the cells of all subgroups are from an animal or human.
- Human cells are particu larly preferred, e.g. preferably tumor cells from a human are distinguished from non-tumor cells from a human, e.g. but not necessarily the same human providing the tumor cells.
- the inventive method or the in ventive device allows the isolation of a subgroup in a biologi cal sample for further examination. If a subgroup is lysed, while another subgroup is kept whole, i.e. or unaffected or less effected by electroporation or lysis, the DNA, RNA, proteins, other molecules or organelles of the lysed or electroporated subgroup can be specifically collected for further examination.
- a subgroup is a type of cells or other cellular bodies with essentially the same properties in view of susceptibility to electroporation and/or lysis in an electrical field
- the subgroup can be a particu lar cell type, such as leukocytes or tumor cells, including sus pended or circulating tumor cells.
- a subgroup com prises the same type of cells or other eukaryotic cellular bodies in the biological sample, e.g. leukocytes or erythrocytes.
- the biological sample can be a blood, saliva, urine sample or any other sample containing eukaryotic cellular bodies.
- the method of targeted electroporation and/or lysis of eukaryotic cellular bodies in a biological sample preferably with at least two subgroups of eu karyotic cellular bodies, wherein each subgroup has a different susceptibility to electroporation and/or lysis in electric fields, comprises the following steps:
- the biological sample to an electric field in the chamber, wherein the electric field is generated by at least two electrodes which are coated with a dielectric material with a relative permittivity greater than 3.9, preferably greater than 9, more preferably 60 or more, and
- the electric parameters of the electric field such as the field strength, the frequency or the wave form so that eu karyotic cellular bodies are electroporated or lysed, prefera bly so that the subgroups are differently affected by said electric field for electroporation and/or lysis.
- the present invention allows the specific lysis and/or electroporation of a subgroup, even only one subgroup, of eukaryotic cellular bodies within a sample with at least two subgroups of eukaryotic cellular bodies.
- the cells or cellular bodies of a subgroup essentially have the same susceptibility to electroporation and/or lysis.
- a subgroup usually refers to cells or cellular bodies of the same type, e.g. leukocytes or erythrocytes.
- sub groups can also be defined as subtypes of cells, for example T- lymphocytes and other lymphocytes, which both belong to leuko cytes.
- T-lymphocytes would represent the first subgroup
- other lymphocytes would represent the second subgroup.
- the second subgroup could be further divid ed into several subgroups, if needed. From this it can be con cluded, the definition of subgroups strongly depends on the ap plication. Surprisingly, it has been found that by selecting particular electric field parameters, eukaryotic cellular bodies can be specifically electroporated or lysed in the electrode as sembly of the present invention.
- the subgroups can be distinguished by their susceptibility to electroporation and/or lysis.
- the different susceptibilities to electroporation of cells depend on the electrical parameters of the applied electrical field, such as the field strength, the pulse frequency of the electric field, the wave form of pulses of the electric field or the exposure time of the subgroups to the electrical field.
- the generated electrical field can be an electrical field with a constant field strength over time. How ever, in a preferred embodiment, the electrical field changes over time or alternates in pulses, in particular with a given wave form, for example a rectangular or sinusoidal wave form of electric field pulses.
- the specific nature of the electric parameters (strength, potential (voltage) , pulse frequency, shape, duration, pulse amount or exerted energy on the cells by the electric field) not only depends on the subgroup of bodies that are differently af fected by the electric field.
- the absolute values also depend on the shape of the chamber - in particular the distance of the electrodes, the thickness of the dielectric material and the me dium in which the bodies are suspended. Different dielectric constants in the medium, different salt types etc. further af fect the efficiency of the electric field so that the electric parameters need adjustment.
- Effects of electroporation and lysis can be easily monitored by e.g. determining leakage of a marker that leaks out of or into the cells when the electric field is applied.
- the following description of parameters are preferred parameters within which cellular bodies can usually be distinguished due to their susceptibility to the electric field.
- the electroporation of cells or other cellu lar bodies of a subgroup depends on the electrical field
- the strength of the applied electrical field which in the present invention lies between 50 V/cm and 50 kV/cm, preferably the ap plied electrical field is from 100 V/cm to 30 kV/cm, from 500 V/cm to 20 kV/cm, or 1 kV/cm to 10 kV/cm, preferably from 2 kV/cm to 5 kV/cm.
- Further preferred is a range of 50 V/cm to 400 V/cm (especially for re versible electroporation) .
- the subgroups may also be distinguished by their sensi tivity to the frequency or the wave form of the applied electri cal field.
- the subgroups may also be distinguished by their sen sitivity to the duration of the exposure time they are exposed to the electrical field.
- combinations of these elec trical parameters e.g. electrical field strength, exposure time and frequency, can be used to distinguish the subgroups more precisely.
- These disclosed ranges for the applied electrical field may also represent ranges for the electrical field in the sample, especially cells or cellular bodies in the sample. Re versible electroporation may be used to transfer compounds into cells, e.g. transfection.
- the ratio of the potential difference between the electrodes and the distance between the electrodes is preferably in the range of 500 V/cm to 50 kV/cm, especially preferred between 50 V/cm and 50 kV/cm, preferably the applied electrical field is from 100 V/cm to 30 kV/cm, from 500 V/cm to 20 kV/cm, or 1 kV/cm to 10 kV/cm, preferably from 2 kV/cm to 5 kV/cm. Especially preferred is a range of 5 kV/cm to 8 kV/cm or 25 kV/cm to 50 kV/cm (especially for lysis) . Further preferred is a range of 50 V/cm to 400 V/cm (especially for reversible electroporation) . This ratio is easier to determine than the field strength - any may be a preferred parameter to character ize the invention.
- the electric field is generated by at least two electrodes which are coated with electrically non-conductive dielectric material.
- the coating at least covers the surface of the electrodes that faces the interior of the chamber. By consequence, it protects the conductive part of the electrode that is behind the coating from contact with the sam ple.
- the dielectric material is solid.
- the dielectric coating is considered to be part of the electrodes.
- the electrodes comprise an electrically conductive part, preferably a metallic part, and a dielectric coating.
- the distances between the electrodes actually refers to the distance between the coatings.
- the electrodes form a part of the chamber' s periph ery.
- the electrodes can be a part of or form an inner surface of the chamber, which inner surface is in contact with the biologi cal sample.
- the electrodes can be essentially flat, flush with the rest of the inner surface or form a structure of any kind.
- the electrodes protrude from the cham ber' s inner surface or form a recess of the chamber' s inner sur face so as to provide a homogenous electric field between the electrodes.
- the electrodes are sheets that are par allel to each other.
- the electrodes are located outside the chamber. However, it is important that the generated electric field penetrates the chamber and hence the sample.
- the electrodes are disposed essentially opposite to each other. In a preferred em bodiment, the electrodes are essentially flat and form two oppo site inner surfaces of the chamber.
- the electrodes are coated with the dielectric material, such that only the dielectric ma terial is in contact with the sample. Electrical current flow would lead to unfavourable side effects, such as (Joule) heating of the sample, electrolyse, pH changes or electrochemical pro Grandes which might negatively influence the sample. These side effects would interfere with the actual electroporation and/or lysis process and thereby aggravate specific electroporation and/or lysis.
- the dielectric material has a relative permittivi ty greater than 3.9, preferably greater than 9, more preferably 60 or more.
- such a dielectric material is re ferred to high-k materials. Even higher relative permittivity is favourable, preferably 80 and higher. In particular preferred embodiments, the permittivity is from 3.9 to 20000, preferably 8 to 10000, in particular preferred 10 to 5000, or 50 to 1000, or even 40 to 500.
- dielectric material any suitable dielectric material can be used, e.g. Titanium dioxide T1O2, Silicon dioxide S1O2, Barium titanate, strontium titanate, aluminium oxide or Ni obium pentoxide, etc.
- a dielectric material with semiconductive properties is used, so as to form a Schottky- diode between the metallic part of the electrode and the dielec tric material.
- Titanium dioxide T1O2 is an example for such a ma terial.
- the dielectrical material can be a thin layer on the elec trodes.
- the material for the electrodes can be copper, gold, silver, platinum, titanium, aluminium, carbon or any other con ducting material.
- the inventive chamber for receiving the biological sample can be a cavity or a flow-through chamber. If the chamber is a flow-through chamber, the chamber has at least one inlet and one outlet for the sample. Thus, the chamber can be referred to as channel. Of course, several chambers or channels can be provid ed. If the chamber is a channel, electroporation and/or lysis can be performed while the sample is flowing through the chan nel.
- the biological sample can be a blood sample, a saliva sample, a urine sample or any other sample that contains eukaryotic cellular bodies. During electroporation and/or lysis, compounds can be added to the biological sample.
- a subgroup can be loaded with markers, stainings or DNA while another subgroup remains unloaded.
- the dielectric material has a thickness below 1 pm, preferably in the range of 50 nm to 850 nm, more preferably in the range of 100 nm to 750 nm or in the range of 150 nm to 500 nm, especially preferred 200 nm to 400 nm. Also larger ranges are possible, e.g. 600 nm to 2000 nm or 700 nm to 1000 nm or 750 nm to 800 nm. Due to the small thickness of the dielectric material, which is coated on the electrodes, the potential drop along the dielec tric material is kept low. Thus, the use of high electrical po tential differences, which entail negative side effects, is avoided. Furthermore, the whole assembly can be realized as mi cro fluidic structure.
- the distance between the electrodes can be below 1 mm, preferably below 550 pm, more preferably below 100 pm or even below 50 pm, but greater than 5 pm.
- the distance between the electrodes is at 5 pm to 1 mm, prefera bly 10 pm to 800 pm, or 20 pm to 700 pm, or 30 pm to 600 pm or 40 pm to 550 pm, or 50 pm to 90 pm or 60 to 85 pm.
- Further pre ferred distances between the electrodes are 120 pm to 2 mm, 150 pm to 1750 pm, 250 pm to 1500 pm, 550 pm to 1200 pm, 600 pm to 1 mm or any combination of these ranges. This facilitates the im plementation of the inventive method in a handheld device since small potential differences can be easily achieved by means of batteries. Additionally, due to the small distances between the electrodes, homogeneity of the electrical field is enhanced.
- the potential difference between the electrodes is in the range of 1 V to 100 V, prefera bly in the range of 5 V to 80 V or even 7 V to 70 V, more pref erably in the range of 10 V to 55 V, 11.8 V to 45 V, 12 V to 40
- the electric field is a periodic field with a frequency in the range of 0.1 Hz to 10 kHz, preferably in the range of 10 Hz to 1 kHz or even 20 Hz to 1 kHz, especially 50 Hz to 900 Hz, or 100 Hz to 800 Hz, or 150 Hz to 700 Hz or 200 Hz to 600 Hz, wherein the wave form of the electric field is prefera bly a square wave, a sinusoidal wave or at least one pulse. Fre quencies around 100 HZ, such as 80 Hz to 200 Hz are especially preferred.
- the periodic field can be unipolar or bipolar. Fur ther, the field can be alternating periodically.
- the pulses are preferably unipolar, with a determined on-time and a determined off-time.
- the pulses are rectangular.
- the pulses are exponential decay pulses, wherein the rising edge has a step form, i.e. a vertical slope with a very short rise time, and the falling edge is exponen tially falling, i.e. has a fall time longer than the rise time, such as multiple times longer, e.g. 5 times, 10 times, 20 times, 100 times or more or any range between these values.
- the wave form can also have a positive or negative offset.
- the wave form can also be cut off partially. Any other methods for shaping the wave form can be used, such as pulse-width modu lation.
- the exposure time of the biological sample lies in the range of 1 second to 20 seconds, preferably in the range of 2 seconds to 12 seconds, more preferably 3 to 7 seconds or essentially 6 seconds. If electroporation should be conducted and the exposure time is too long, too many cells may die during the process.
- the number of pulses is kept in the range from 1 to 40000 or 10 to 30000, preferably in the range from 50 to 20000, more preferably in the range from 100 to 10000 or 150 to 8000, in particular preferably in the range from 200 to 8000 or 400 to 7000.
- Other favourable ranges, particularly for delivering compounds into cellular bodies are 1 to 100 pulses, 1 to 70 pulses, 1 to 40 pulses, 1 to 15 pulses or 1 to 7 pulses.
- Other favourable ranges are 1000 to 20000 pulses, 1000 to 15000 pulses, 1000 to 10000 pulses, 1000 to 5000 pulses or 1000 to 3000 pulses.
- the fre quency at which the above mentioned number of pulses are applied is in the range of 10 Hz to 5 kHz, preferably, 100 Hz to 2 kHz, more preferably 800 Hz to 1.5 kHz.
- Pulse refers to one monopo lar excitation to a maximum (such as at the indicated poten tial/voltages discussed above) that drops towards to baseline excitation, such as one-half sinusoid period or one square pulse, or one exponentially falling pulse.
- the sample can be filtered for concentrating cellular bodies of a subgroup af ter lysis and/or electroporation.
- the chamber can comprise a filter to concentrate and purify particles or cells un-/affected from electroporation and/or lysis.
- DNA or RNA material from the cellu- lar interior is set free, in a preferred embodiment, after lysis of a subgroup, organelles and/or biomolecules from said sub ⁇ group, preferably genetic material such as DNA or RNA or pro ⁇ teins, are collected. Other biomolecules are lipids of the lysed cells .
- Electroporation can be used to collect material from or in troduce material into the cellular body.
- material can be compounds of the cell, such as RNA or proteins.
- Material intro ⁇ quizged into the cells is usually nucleotides, especially RNA or DNA or small molecule compounds of a size of up to 1 kDa .
- markers, labels or dyes are introduced into the cell, such as radiolabels or fluorescent labels.
- an electrical conductivity reduction of the sample below lmS/cm preferably in the range of 10 pS/cm to 800 pS/cm or 60 pS/cm to 560 pS/cm, by means of ionic exchange, transversal diffusion, filtering, dilution, buffer exchange or electrophoretic separa ⁇ tion is conducted.
- the conductivity reduction can be conducted in a separate or in the same device as the inventive method.
- the alkali metal ions are with a halogen counterion, such as CIA
- the concentration of the added molecules or salts lies in the range of 0.001 mM to 100 mM, preferably 0.01 mM to 50 mM, more preferably in the range of 0.05 mM to 10 mM for Li, Na, such as LiCl, NaCl, MgCA, H X PC>4 or combinations thereof.
- mM refers to the unit millimolar. In case of combinations, these indicated concentrations refer to a sum concentration of these (alkali) metals combined.
- flow cytometry For the purpose of examining cells or cellular bodies, it is advantageous if prior to and/or after exposing the biological sample to the electrical field, flow cytometry is conducted.
- cells are passed by an electrical potential or a light beam and thereby lead to different measura ble effects depending on the size, shape or colour of the cells.
- it can be useful to load the cells of a certain subgroup of the biological sample with markers, stain- ings or other compounds prior to flow cytometry.
- the biological sample prior to flow cytometry, can be exposed to the electrical field in the inventive chamber for electroporation and loaded with markers, stainings or other compounds.
- the biological sample can be transferred in the inventive chamber for exposing the bi ological sample to the electrical field.
- Both embodiments (elec tric field treatment prior and after flow cytometry) can be com bined.
- two inventive chambers with electrodes are used, wherein the chambers are in fluid connec tion with the flow cytometry unit.
- One of the chambers is ar ranged upstream to the cytometry device and the other chamber is arranged downstream to the cytometry device. This arrangement can be accommodated in a single device or separated from each other.
- the sample is loaded with markers, stainings or other compounds in the first chamber, then transferred to the flow cytometry unit via fluid connection.
- the sample is transferred to the second chamber via fluid connection.
- the identified subgroups can be electroporated or lysed in the second chamber.
- the chamber comprises two planar electrodes which are spaced apart with a distance of 81.3 pm.
- the potential difference can be calcu lated by means of a multiplication of the distance by the elec trical field strength.
- the electrical field strength can be calculated from the distance and the potential difference between the electrodes (potential difference divided by distance) .
- the elec trical field strength can be determined (neglecting the poten tial drop in the coating of the electrodes by approximation) . Therefore, the electrical field strength can be unambiguously exchanged with the specification of the distance between the electrodes and the potential difference and vice versa without any restriction.
- the distance could also be greater or smaller than 81.3 pm as discussed above.
- the electric field has a field strength of at least 2.2 kV/cm, preferably of at least 2.5 kV/cm, more preferably 3.0 kV/cm, but not more than 6 kV/cm or even not more than 4.5 kV/cm.
- the electric field can be a periodic field and the frequency can be in the range of 10 Hz to 1 kHz, preferably 50 Hz to 200 Hz.
- the wave form of the electrical field is an alter nating square wave or unipolar rectangular pulses and the fre quency is preferably about 100 Hz.
- the exposure time of the sam ple in the electrical field lies between 12 seconds and 0.5 sec onds. Assuming that the distance between the electrodes is 81.3 pm, the corresponding potential difference is at least about 17.8 V, preferably at least about 20 V, more preferably about 25 V, but not more than about 50 V or even not more than 36.6 V.
- leukocytes can be isolated from erythrocytes.
- the erythrocytes are thereby lysed.
- the electric field has a field strength of at least essentially 3.0 kV/cm, preferably 4.5 kV/cm.
- the electric field can be a periodic field and the frequency can be in the range of 10 Hz to 500 Hz, preferably 100 Hz to 200 Hz.
- the wave form of the electric field is an alternating square wave or unipolar rectangular pulses and the frequency is preferably about 100 Hz.
- the exposure time of the sample in the electrical field lies between 12 seconds and 0.5 seconds. Assum ing that the distance between the electrodes is 81.3 pm, the corresponding potential difference is at least essentially 25 V, preferably 36.6 V, but preferably not more than 45 V.
- the step of choosing the electric parameters of the electric field is easy within the above described parameters.
- these parame ters can be varied to identify a parameter combination that is able to lyse or electroporate cells, in particular, so that se lected subgroups of cells are differently affected by said elec tric field for electroporation and/or lysis.
- the choosing step may include a brief screening step. E.g. it is possible to pro vide two subgroup of cells and select a pulse shape and pulse frequency and treatment duration, e.g. within the ranges de scribed herein. Then a potential is varied, e.g.
- the pulse frequency and treatment duration shall be varied and again the voltage is tested for a value of different susceptibility between the sub groups of cells. This simple procedure can be repeated for all electric parameters of the electric field as described herein.
- the electric field has a field strength of at least 1.8 kV/cm, preferably 2.4 kV/cm.
- the electric field can be a periodic field and the fre quency can be in the range of 10 Hz to 1000 Hz, more preferably 100 Hz to 200 Hz.
- the wave form of the electrical field is an alternating square wave or unipolar rectangular pulses and the frequency is preferably about 100 Hz.
- the expo sure time of the sample in the electrical field lies between 12 seconds and 0.5 seconds.
- the corresponding potential difference is at least about 14.6 V, preferably at least about 20 V, more preferably about 19.6 V, but preferably not more than 45 V.
- Jurkat T lymphocytes can be cancerous Leukocytes.
- the above method can be described as a method of targeted electroporation and/or lysis of eukaryotic cellular bodies in a biological sample with at least a first and a second group or subgroup of eukaryotic cellular bodies, each group or subgroup having an electroporation and/or a lysis rate, wherein the biological sample is transferred into a chamber and exposed to an electric field in the chamber which is generated by at least two electrodes which are coated with a dielectric material with a relative permittivity greater than 3.9, preferably great er than 9, more preferably 60 or more, wherein for isolating and/or delivering a compound into the cells of the first group or subgroup the electric parameters such as the field strength, the frequency or the wave form of the electric field are chosen to conduct electroporation and/or lysis the first group or sub group with a different electroporation and/or a lysis rate than the second group or subgroup.
- the invention relates to a device, pref erably handheld device, for targeted electroporation and/or ly sis of eukaryotic cellular bodies in a biological sample, com prising at least one chamber for receiving the biological sample and at least two electrodes for generating an electric field in the chamber, which electrodes are coated with a dielectric mate rial with a permittivity greater than 3.9, preferably greater than 9, more preferably 60 or more, wherein the distance between the electrodes is below 1 mm, preferably below 550 pm, more preferably below 100 pm, but more than 5 pm, wherein the device further comprises an adjustment unit that enables the adjustment of electric parameters of the electric field such as the field strength, the frequency or the wave form.
- the distance between the electrodes is kept below 1 mm.
- the distance between the electrodes is at 5 pm to 1 mm, preferably 10 pm to 800 pm, or 20 pm to 700 pm, or 30 pm to 600 pm or 40 pm to 550 pm, or 50 pm to 90 pm or 60 to 85 pm.
- Further preferred distances between the electrodes are 120 pm to 2 mm, 150 pm to 1750 pm, 250 pm to 1500 pm, 550 pm to 1200 pm, 600 pm to 1 mm or any combination of these ranges.
- the potential difference between the elec trodes can be kept low and the homogeneity of the electrical field is improved.
- the chamber can be a cavity as well as a flow-through chamber with an inlet and an outlet. If the chamber is a flow-through chamber, it is also referred to as channel. It is important to note that parts of the electrodes that are in contact with the biological sample in the filled state of the chamber are coated with the electrically non-conductive dielec tric material. As the device is intended for implementing the above described method, reference is made to the above remarks.
- the dielectric material has a thickness below 1 pm, preferably in the range of 50 nm to 850 nm, more preferably in the range of 100 nm to 750 nm or in the range of 150 nm to 500 nm, especially preferred 200 nm to 400 nm. Also larger ranges are possible, e.g. 600 nm to 2000 nm or 700 nm to 1000 nm or 750 nm to 800 nm.
- the chamber forms a part of an air displacement pi pette, particularly a part of a tip of such a pipette, wherein the pipette has suction and pressure means for creating a reucked pressure within the pipette.
- Air displacement pipettes are also known as micropipettes.
- the tip is considered to form a part of the pipette, even when the tip is exchangeable.
- the electrodes can be arranged in the longitudinal direction of the tip. By means of the suction and pressure means the sample can be sucked into the tip and then be exposed to the electrical field within the tip. The reduced pressure is thus directly or indirectly generated within the tip.
- the suction and pressure means are preferably formed by a piston, which can be actuated by hand or by an actuator, such as an electrical actuator.
- the chamber can have a size in the range of 0.001 to 30 ml, prefera bly 0.01 to 25 ml, or 0.1 ml to 20 ml, 0.2 ml to 15 ml, 0.5 ml to 12 ml, 1 ml to 10 ml or 1 ml to 5 ml .
- the sample can be released again by means of the suction and pressure means. It is preferred when the tip is removable from the pipette and hence exchangeable.
- all units of the device including the adjustment unit and batteries, are integrated into the pipette.
- the adjustment unit of the device is separated from the pipette.
- the electrodes can be connected to the adjustment unit via electrical wires.
- the electrodes of the chamber form an inner surface of the chamber and lie opposite to each other.
- the chamber has a longitudinal extension and the electrodes are ring electrodes that encircle the chamber from outside, wherein the electrical field penetrates the cham ber.
- the device is an air displacement pi pette with a suction and pressure means for creating a reduced pressure in a receiving chamber, wherein receiving chamber is suitable for receiving a biological sample, wherein the chamber has at least two electrodes for generating an electric field in the chamber, which electrodes are each on an inner surface of the chamber and which electrodes are coated with a dielectric material with a relative permittivity greater than 3.9, prefera bly greater than 9, more preferably 60 or more, wherein the dis tance between the electrodes is below 1 mm, preferably below 550 pm, more preferably below 100 pm.
- the device further is connected to or comprises an adjustment unit that enables the adjustment of electric parameters of the electric field such as electric field strength, electric pulse frequency or electric pulse wave form of the electric field.
- Air displacement pipettes are also known as micropipettes.
- the suction and pressure means are formed by a pis ton, which can be actuated by hand or by an actuator, such as an electrical actuator.
- the chamber is accommodated within a tip of the pipette.
- the electrodes can be arranged in the longitudinal direction of the tip.
- the tip is preferably ex changeable.
- the tip is considered to form a part of the pipette, even when the tip is exchangeable.
- the chamber can have a size in the range of 0.001 to 30 ml, preferably 0.01 to 25 ml, or 0.1 ml to 20 ml, 0.2 ml to 15 ml, 0.5 ml to 12 ml,
- all units of the device are inte grated into the pipette.
- the power supply e.g. batteries
- the adjustment unit of the device is separat ed from the pipette.
- the electrodes can be connected to the ad justment unit via electrical wires.
- the adjustment unit directly supplies the electrodes with voltage to generate the electrical field .
- the invention provides a method of tar geted electroporation and/or lysis of cellular bodies in a bio logical sample, comprising the following steps:
- the electric field is generated by at least two electrodes which are coated with a dielectric material with a relative permittivity greater than 3.9, preferably greater than 9, more preferably 60 or more, wherein the dis tance between the electrodes is below 1 mm, preferably below 550 pm, more preferably below 100 pm, and
- the electric parameters of the electric field such as the field strength, the frequency or the wave form so that the cellular bodies are lysed and/or electroporated.
- This method may be used for lysis of cells or transfection.
- the biological sample is transferred into the tip of the pipette and the parameters are chosen such that the cells are still alive after exposure to the field (reversible electro poration) .
- material or a component that may be added to the biological sample is transferred into the cells.
- lysis can be conducted as well.
- the suction and pressure means are formed by a piston, which can be actuated by hand or by an actuator, such as an electrical actuator.
- the chamber can have a size in the range of 0.001 to 30 ml, preferably 0.01 to 25 ml, or 0.1 ml to 20 ml, 0.2 ml to 15 ml, 0.5 ml to 12 ml, 1 ml to 10 ml or 1 ml to 5 ml .
- a size in the range of 0.001 to 30 ml, preferably 0.01 to 25 ml, or 0.1 ml to 20 ml, 0.2 ml to 15 ml, 0.5 ml to 12 ml, 1 ml to 10 ml or 1 ml to 5 ml .
- the electrodes are flat sheets or metallic mate rial (with a passivation layer as described above) .
- the surface facing the chamber that is able to be in contact with the sample in the chamber is preferably 50 mm 2 to 1000 mm 2 , especially pre ferred 75 mm 2 to 750 mm 2 , 100 mm 2 to 300 mm 2 .
- the pipette tip or cham ber has not more than two electrodes for treatment of the sam ple.
- the chamber is preferably rectangular with the electrodes provided in parallel arrangement therein, forming the rectangu lar space.
- the electrodes in this size are preferably adapted for smaller voltages, such as 1 V to 45 V or preferably 5 V to 40 V.
- the electrodes are preferably spaced so as to cover the entire chamber.
- the chamber volume between the electrodes is sub stantially equal or bigger than the volume sucked in to the pi pette tip and/or the volume of the piston of the pipette that defines said suction volume per tip.
- the electrodes extend substantially to the chamber opening at the tip of the pipette .
- a method of targeted electroporation and/or lysis of eukary otic cellular bodies in a biological sample with at least two subgroups of eukaryotic cellular bodies, wherein each subgroup has a different susceptibility to electroporation and/or lysis in electric fields comprising the following steps:
- the biological sample to an electric field in the chamber, wherein the electric field is generated by at least two electrodes which are coated with a dielectric material with a relative permittivity greater than 3.9, preferably greater than 9, more preferably 60 or more, and
- the biological sample to an electric field in the chamber, wherein the electric field is generated by at least two electrodes which are coated with a dielectric material with a relative permittivity greater than 3.9, preferably greater than 9, more preferably 60 or more, and
- the electric parameters of the electric field such as the field strength, the frequency or the wave form so that eu karyotic cellular bodies in a biological sample are affected by said electric field for electroporation and/or lysis.
- the dielectric material has a thickness below 1 mpi, preferably in the range of 50 nm to 650 nm, more preferably in the range of 100 nm to 500 nm.
- the wave form of the electric field is preferably a square wave, a sinusoidal wave or at least one pulse per peri od .
- the elec tric field has a field strength of at least 2.2 kV/cm, prefera bly of at least 2.5 kV/cm, more preferably 3.0 kV/cm, but not more than 6 kV/cm or even not more than 4.5 kV/cm.
- the electric field has a field strength of at least essentially 3.0 kV/cm, preferably 4.5 kV/cm.
- the electric field has a field strength of at least 1.8 kV/cm, preferably 2.4 kV/cm.
- Method for manufacturing a device, preferably handheld de vice, particularly an air displacement pipette, for targeted electroporation and/or lysis of eukaryotic cellular bodies wherein at least two electrodes are coated with a layer of die lectric material with a permittivity greater than 1, preferably greater than 3.9, more preferably 60 or more, wherein the elec trodes are arranged such that the distance between the elec trodes is below 1 mm, preferably below 550 pm, more preferably below 100 pm, but greater than 5 pm. 17.
- Device suitable for targeted electroporation and/or lysis of eukaryotic cellular bodies in a biological sample, comprising at least one chamber for receiving the biological sample and at least two electrodes for generating an electric field in the chamber, which electrodes are coated with a dielectric material with a permittivity greater than 1, preferably greater than 3.9, more preferably 60 or more, wherein the distance between the electrodes is below 1 mm, preferably below 550 pm, more preferably below 100 pm, but greater than 5 pm, wherein the device further comprises an adjustment unit that enables the adjustment of electric parameters of the electric field such as the field strength, the frequency or the wave form.
- the chamber forms a part of an air displacement pipette, particularly a part of a tip of such a pipette, wherein the pipette has suction and pressure means for creating a reduced pressure within the pipette such that the biological sample is transferred into the pipette.
- An air displacement pipette device with a suction and pres sure means for creating a reduced pressure in a receiving cham ber wherein the receiving chamber is suitable for receiving a biological sample, wherein the chamber has at least two elec trodes for generating an electric field in the chamber, which electrodes are each on an inner surface of the chamber and which electrodes are coated with a dielectric material with a relative permittivity greater than 3.9, preferably greater than 9, more preferably 60 or more, wherein the distance between the elec trodes is below 1 mm, preferably below 550 pm, more preferably below 100 pm.
- the device further is connected to or comprises an adjustment unit that enables the adjustment of electric parameters of the electric field such as electric field strength, electric pulse frequency or electric pulse wave form of the electric field.
- the dielectric ma terial has a thickness below 1 pm, preferably in the range of 50 nm to 650 nm, more preferably in the range of 100 nm to 500 nm.
- the distance be tween the electrodes is below 1 mm, preferably below 550 pm, more preferably below 100 pm or even below 50 pm, but greater than 5 pm, and the electric field strength is in the range of 500 V/cm to 50 kV/cm, preferably 1 kV/cm to 10 kV/cm, more pref erably 2 kV/cm to 5 kV/cm.
- the electric field is a periodic field with a frequency in the range of 0.1 Hz to 10 kHz, preferably in the range of 10 Hz to 1 kHz or even 50 Hz to 1 kHz, wherein the wave form of the electric field is prefer ably a square wave, a sinusoidal wave or at least one pulse per period .
- the device of any one of 17 to 23 wherein the chamber has a size of 0.001 to 30 ml, preferably 0.01 to 25 ml, or 0.1 ml to
- sivation is equal to coating and that "being passivated" means being coated with dielectric material.
- Fig. 1 shows a comparison of lysis curves of diluted whole blood and leukocyte suspension.
- Fig. 2 refers to the enrichment of leukocytes in a leuko cyte-spiked whole blood dilution.
- Fig. 3 shows microscopy images of leukocyte-spiked diluted blood before and after application of an electric field.
- Fig. 4 shows the results of flow cytometric analysis for isolated leukocytes with no field (images A to D) and after 40 V square waves applied at 100 Hz (images E to H) . Histograms show cell viability via Calcein-AM fluorescence (images A, E) , CD45 fluorescence shows cells other than erythrocytes (images B, F) and cells stained for CD34 account for the fraction of HSCs (im ages C, G) . Images D and H refer to gate statistics and logic.
- Fig. 5 shows the voltage-dependence of Jurkat T lymphocyte cells and healthy leukocyte lysis. Data points represent the mean of four technical replicates, error bars indicate standard deviation .
- Fig. 6 shows the frequency dependent viability of Leukocytes (image A) and Jurkat T lymphocytes (image B) .
- Conductivity val ues refer to measurements before and after the experiment. Data points represent the mean of three technical replicates, error bars indicate standard deviation.
- Fig. 7 concerns the selective elimination of Jurkat T lym phocyte population spiked to lymphocyte suspension.
- Jurkat T lymphocytes were incubated with Calcein-AM prior to data acqui sition and have high FITC fluorescence.
- Image A shows mixed cell population without electric field application.
- Image B shows the mixed cell population after application of 20 V square wave pulses at 50 Hz.
- Image C shows the event statistics showing per centage of respective events.
- Conductivity: 97 pS/crn. Events: 10 000; N 3.
- Fig. 8 shows the Voltage-dependent viability of MCF-7 breast cancer cells and healthy leukocyte lysis. Data points represent the mean of four technical replicates, error bars indicate standard deviation.
- Fig. 9 refers to frequency dependent viability of Leukocytes (image A) and MCF-7 breast cancer cells (image B) .
- Conductivity values refer to measurements before and after the experiment.
- Fig. 11 shows the inventive device.
- Image A shows a multi functional pipette as inventive device with attached pipette tip comprising electrodes.
- Image B shows a pipette tip connected to a 20 pi pipette with electrical circuit connection.
- Image C shows a transparent version of the pipette tip showing microflu idic properties.
- Image D gives a cross sectional view of the chamber.
- Image D gives a cross sectional view of the chamber in an alternative embodiment, wherein 8 electrodes are located in the chamber's circumferential surface.
- Image D refers to measurement of cell-free DNA after lysis of HER cells in a pipette tip.
- Image E refers to the measurement of cell-free RNA after lysis of HER cells in the pipette tip.
- Fig. 13 refers to 293T cell lysis and permeabilization after exposure to an electrical field.
- Image A shows 293T cell lysis and permeabilization after exposure to decreasing pulse number of 40 V square waves
- Image B shows 5 square wave pulses of in creasing voltage
- Image C shows an increasing number of 15V square wave pulses.
- D Technical replicates of 293T transfec tion with pTurboRFP plasmid upon exposure to 10 exponential de cay pulses of 40V at 50 Hz and 1000 exponential decay transfer pulses of 15 V at 50 Hz. Bars show percentage of cells express ing RFP 48 hours after treatment as detected by fluorescence mi croscopy .
- Fig. 14 shows a flow cytometry application in connection with the inventive method and device for specific electro
- Fig. 15 shows conductivity dependent whole blood lysis in a lab-scale flow-through prototype. Data points represent the mean of three technical replicates, error bars indicate standard de viation
- Fig. 16 shows: (a) Lab-scale flow-through prototype compris ing anodic oxidized titanium electrodes, (b) I/V plot during ap plication of 8 V 50 Hz square wave, (c) Lysis efficiency of whole blood cells when applying 8 V, 50 Hz square wave with 100 m ⁇ /min to a 1:100 whole blood dilution.
- Fig. 17 shows: (a) Comparison of lysis curves of diluted whole blood and leukocyte suspensions shows different behavior in the electric field. Frequency-dependence of whole blood cells (mainly erythrocytes) at 18 V (b) and leukocyte lysis at 40 V (c) . Data points represent the mean of three technical repli cates, error bars indicate standard deviation, s is the suspen sion conductivity.
- Fig. 18 shows the Enrichment of leukocytes (Hoechst+) in a leukocyte-spiked whole blood dilution. Porated cells
- Fig. 19 shows: (a) Comparison of lysis curves of leukocyte cell suspensions and Jurkat T cell suspensions shows different behavior in the electric field. Frequency-dependent lysis of leukocytes at 40 V (b) and Jurkat cells at 18 V (c) . Data points represent the mean of three technical replicates, error bars in dicate standard deviation, s is the suspension conductivity.
- Fig. 20 shows: (a-d) Flow cytometry data from 10,000 events show removal of the distinct Jurkat T lymphocyte population af ter application of an electric field (20 V, 50 Hz, sqAC) . (a)
- Fig. 21 shows: Comparison of lysis curves of leukocyte cell suspensions and MCF-7 cell suspensions dependent on electric field strength. Frequency-dependent lysis of leukocytes at 40 V (b) and MCF-7 cells at 20 V (c) . Data points represent the mean of three technical replicates, error bars indicate standard de viation .
- Fig. 22 shows: Selective elimination of FITC-Antibody stained MCF-7 cells spiked to a leukocyte suspension. Hoechst 33342 viability stain was added prior to flow cytometry data ac quisition.
- (d) Statistics on viability after field application. Conductivity: 100-103 pS/citi. Events: 10 000. N 2
- Fig. 23 shows: Flow cytometry histograms of a first idea gation to use electric fields for the enrichment of hematopoiet ic stem (CD45+CD34+) cells from a leukocyte cell suspension, (a) Sample after passing the lab-scale flow-through prototype with out electric field applied, (b) Sample after passing the lab- scale flow-through prototype with 40 V 100 Hz applied.
- Fig. 24 shows: Comparison of permeabilization and electric field induced lysis of HER (human embryonic kidney) cells using a classical cuvette-based commercial electroporation system (a) and the developed electroporation pipette tip (b) .
- Fig. 25 shows: Occurrence of cell permeabilization by using a large number of pulses at low voltage, termed “Low Energy Mode” (a) and only 5 pulses at higher electric potentials, termed “High Energy Mode” (b) .
- Fig. 26 shows: Change of the pulse setup used for dye transport showing electric discharge (a) to exponential decay pulses to induce a net electrophoretic movement of DNA molecules (b) .
- Fig. 27 shows: Plasmid transfection efficiency of 293T HER cells in electroporation buffers of increasing MgCl2 concentra tion .
- Fig. 28 shows: (a) Electric field induced lysis of HER cells in dependence on buffers comprising different ionic composition at equal conductivities, (b) Transfection efficiency of HER 293T cells in MgCl2 and LiCl buffer using exponential decay pulses and in LiCl buffer using square wave pulses.
- Fig. 29 shows: Electric field induced cell lysis of differ ent cultured cell lines at standardized cell number and buffer conductivity .
- Fig. 30 shows: Investigation of cell-size dependent electric field induced lysis via the alteration of cell size through os motic regulation.
- Cell size is presented as arbitrary forward scatter (FSC, blue) and percentage of cell lysis at 30 V 6000 pulses is shown in red.
- FSC forward scatter
- Fig. 31 shows: Electrical characterization, (a) Leakage cur rent of lab-scale flow-through prototypes filled with different conductive solutions measured by applying a DC current at indi- cated voltage, (b) Diode characteristic measurements using 4 different high-k passivated electrodes ( PEPP1-PEPP4 ) .
- Fig. 32 shows an I/V diagram of non-passivated (grey hash) and thermally passivated titanium electrodes (T1O2, red circles and blue triangles) .
- T1O2 (break down) in blue triangles shows an example of passivation deficiency and dielectric breakdown.
- Example 1 erythrocyte specific lysis for the isolation of leu kocytes
- Fig. 1 shows a comparison of lysis curves of diluted whole blood and leukocyte suspension.
- the abscissa is the potential difference between the electrodes, the ordinate is the viability in per cent.
- Fig. 1 refers to determination of the lysis parameters of erythrocyte- and leukocyte populations separately prepared from whole blood.
- the electrical field has a frequency of 100 Hz and the wave form is a square wave ac signal.
- a specific field ef fect acting on erythrocytes can be observed in the 18 V elution with a reduction in cell viability of 77.9 %.
- At 20V only 2.8 % of erythrocytes remain intact while the leukocyte population is unaffected with 95.6 % viability.
- Complete erythrocyte lysis is achieved at 25 V (0.6 % viable) . Since this refers to a whole- blood dilution it also contains a remaining leukocyte popula tion.
- a spike-in suspension of whole-blood and isolated leukocytes was prepared for a final cell ration of 1:1.
- Conductivity was adjusted to 220 pS/cm and voltage set to 25 V with all other parameters unchanged.
- a dou ble stain of Propidium Iodide (PI) and Hoechst 33342 was used to assess any change in the membrane integrity of the remaining leukocyte population. Both dyes bind DNA and as such will only visualize the nucleus of leukocytes.
- PI Propidium Iodide
- Hoechst 33342 was used to assess any change in the membrane integrity of the remaining leukocyte population. Both dyes bind DNA and as such will only visualize the nucleus of leukocytes.
- the positive popula tion represents leukocytes with compromised membranes.
- Hoechst 33342 is added subsequently to allow for clear distinction of leukocytes from erythrocytes.
- 20% of the total 50% leukocytes are PI positive, indicating some kind of membrane aberration due to the standard multistep protocol for the isolation of leukocytes.
- Af ter field application and selective erythrocyte lysis the frac tion of intact leukocytes remains equal (40 % of 100 %) . This indicates that the applied field has no impact on membrane in tegrity in the surviving population.
- Microscopy images B to D show leukocyte-spiked diluted blood before (0V) and after the application of an electric field (25V, 100 Hz, square wave AC) .
- B shows a merge of fluorescence and brightfield image.
- Hoechst-stained leukocytes in untreated sus pension appear blue, erythrocytes and other cells from blood ap pear translucent orange.
- Image C shows the enrichment of leuko cytes after field application.
- Image D shows Hoechst-positive leukocytes in blue fluorescence channel.
- Pi-positive cells are visible in the green fluorescent channel.
- the conduc tivity s was 220 - 230 pS/crn.
- Example 2 Lysis of leukocyte sub-populations leads to enrich ment of CD34-positive cells
- HSCs Hematopoetic stem cells
- Fig. 4 shows the effects of applying high- energy square wave pulses of 40 V at 100 Hz to isolated leuko cytes with respect to the fraction of CD34 HSCs.
- Calcein-AM is used to determine cell viability and CD45, also referred to as leukocyte common antigen, should be expressed on all leukocytes.
- ELB Eryth rocyte lysis buffer
- ELB 10 ml ELB is added to 1ml of whole blood, incubated lOmin RT and inverted repeatedly. The suspension is centrifuged at 500g for lOmin (Eppendorf 5430; Rotor: F-35-6-30) . These steps are repeated until cell pellet is white (void of erythrocytes) . It is then washed twice with sucrose + PBS solution set to
- Leukocyte precursor enrichment was assessed by co-staining of samples with Ca-AM, Alexa Fluor 700 conjugated CD45R (B220) Monoclonal AB (RA3-6B2, Thermo Fischer, 56-0452-82) and PE con jugated CD34 Monoclonal Antibody (4H11, Thermo Fischer, 12-0349- 42) for 30 min RT in the dark after ECLU field application.
- B220 Alexa Fluor 700 conjugated CD45R
- RA3-6B2 Thermo Fischer, 56-0452-82
- PE con jugated CD34 Monoclonal Antibody 4H11, Thermo Fischer, 12-0349- 42
- Antibody compensation was performed with VersaComp An tibody Capture Beads (Beckmann Coulter, B22804) and Ca-AM was compensated with stained 293T cells (supplied by the Department of Nanobiotechnology of the University of Natural Resources and Life Sciences, Vienna) . Gating and analysis was performed with Kaluza Analysis Software (Beckmann Coulter) .
- Example 3 Leukemia specific cell ablation in a mixture of healthy blood cells
- Fig. 5 shows viability of Jurkat T lymphocytes, a leukemia cell model, and Leukocytes from a healthy volunteer when sub jected to square wave pulses of increasing magnitude.
- Jurkat T lymphocytes show the highest susceptibility to voltage dependent lysis.
- Square wave pulses of 15 V reduce cell viability of Jurkat cells to 22.6 %.
- Fig. 6 A and B shows frequency dependency of the same cell types at respective field magnitude.
- Fig. 7 A, B shows the flow cytometry data with and without field application to a 1:1 mixture of Jurkat T lymphocyte- and leukocyte suspensions in equally equilibrated buffer.
- 31.6 % of counted events are Calcein-AM positive Jurkat T lymphocytes and 41.7 % are identified as leukocytes by their SSC profile (Fig. 7 A) .
- Fig. 7 B Upon application of 20 V square wave pulses at 50 Hz, ⁇ 0.1 % of the events remain Calcein-AM positive Jurkat T lymphocytes while leukocytes make up 50.7 % of events (Fig. 7 B) . Cellular debris increases from 11.4 % to 26.5 % with field application (Fig. 7 B, C) .
- the claim that Jurkat T lymphocytes undergo lysis instead of losing Calcein-AM fluorescence is sup ported by SSC gating and previous lysis experiments.
- Fig. 7 C shows the average event counts for three technical replicates with standard deviation.
- Example 4 Breast cancer specific cell ablation in a mixture of healthy blood cells
- Fig. 8 shows viability of MCF-7 and Leukocytes when subject ed to square wave pulses of increasing magnitude.
- MCF-7 is a breast cancer cell line broadly used as circulating tumor cell model in research.
- Square wave pulses of 15 V reduce cell via bility of MCF-7 to 30.7 % on average.
- both cancer models (Example 1 and 2) display a higher field sus ceptibility than erythrocytes at the same field strength and conductivity (Fig. 1, 74,3 % viable) .
- Fig. 9 A and B show frequency dependency of the same cell types at respective field magnitude.
- Fig. 10 A-B shows the flow cytometry data with and without field application to a 1:1 mixture of MCF-7 and leukocyte popu lations in buffer.
- MCF-7 cells are stained with FITC-conj ugated antibody for identification. Viability is assessed via Hoechst 33342 staining prior to data acquisition.
- 69.8 % of labelled MCF-7 cells are viable with 91.7 % of leukocytes remaining intact (Fig. 10 A, C) .
- Application of 30 V square wave pulses at 100 Hz reduces the viability of the MCF-7 population to 2.1 % while 68.5 % of leukocytes remain viable (Fig. 10 B, C) .
- Fig. 10 C shows the av erage population counts for two technical replicates with stand ard deviation.
- ELB electroporation buffer
- Jurkat T lymphocytes (supplied by the Department of Nanobi otechnology of the University of Natural Resources and Life Sci ences, Vienna) were cultivated at 37 °C and 5% C02 in RPMI
- Thermo Fisher, 21875091 supplemented with 10% FBS (Thermo Fisher, 10500) and 1% Pen/Strep Antibiotic-Antimycotic (Thermo Fisher, 15240) .
- Jurkat are passaged by centrifugation for 5 min at 400 g (RT, Eppendorf 5430; Rotor: F-35-6-30) .
- MCF-7 were cul tivated at 37 °C and 5% C02 in MEM (Thermo Fisher, 21875091) supplemented with 10% FBS (Thermo Fisher, 10500), 2% L-Glutamin (Thermo Fisher, 25030081), 1% Non-essential amino acids (Thermo Fisher, 11140050) and 1% Pen/Strep Antimycotic-Antimycotic
- MCF-7 are passaged by washing with PBS (lx from stock: Thermo Fisher, 70011044) followed by trypsiniza- tion (0,25%, Thermo Fisher, 25200) for 5 min at 37 °C. Any ster ile protocols were processed in biological safety cabinets. (He- rasafe KS, Class II, Thermo Fisher, 51022488)
- ELB 10 ml ELB is added to 1ml of whole blood, incubated lOmin RT and inverted repeatedly. The suspension is centrifuged at 500g for lOmin (Eppendorf 5430; Rotor: F-35-6-30) . These steps are repeated until cell pellet is white (void of erythrocytes) . It is then washed twice with sucrose + PBS solution set to
- Jurkat T lymphocyte-spiked leukocyte suspension both populations were prepared as described in sections 2.3 and 2.4.
- Jurkat-T lymphocytes in EPB were stained with lOnM Calcein- AM solution (Thermo Fischer, C3100MP) for 60min at 37°C to allow for population tracking in flow cytometry. An aliquot of un stained cells was set aside for flow cytometry negative con trols. Stained Jurkat T lymphocytes are mixed 1:1 for final con centrations of 5x105 each prior to pulse application.
- MCF-7-spiked leukocyte suspension both populations were pre pared as described as above.
- MCF-7 cells in EPB were stained with CD326-FITC-Antibody (1:20) for 30min at 4°C to allow for population tracking in flow cytometry. An aliquot of unstained cells was set aside for flow cytometry negative controls.
- MCF-7 are mixed 1:1 for final concentrations of 5x105 each prior to pulse application. Live-dead discrimination of the MCF-7 population was not possible based on FITC-fluorescence alone. All spike-in samples were stained with lpg/ml Hoechst 33342 solution and incubated 5 min RT prior to data acquisition.
- FACSCanto II Forward scatter (FSC) and side scatter (SSC) thresholds were set to eliminate cell debris from the final readout. 10.000 events were recorded for each parameter. Ca-AM and CD326-FITC parameters were recorded in the FITC channel, Hoechst 33342 staining is recorded in the Pacific Blue channel. No compensation control is included due to lack of fluorescence overlap. Data was gated in Kaluza Analysis Software (Beckmann Coulter) and results plotted in Graphpad Prism 7.
- Example 5 Device for targeted electroporation and/or lysis of eukaryotic cellular bodies in a biological sample with at least two subgroups of eukaryotic cellular bodies
- Fig. 11 shows the inventive device for targeted electro poration and/or lysis of eukaryotic cellular bodies in a biolog ical sample with at least two subgroups of eukaryotic cellular bodies as a pipette.
- the chamber is located within the tip of the pipette, as can be seen in Fig. 11 B.
- Fig. 11 D and Fig. 11 E show two different embodiments of the chamber in a cross sec tional view. 1 refers to the electrodes, while 2 refers to the housing, which is not conductive.
- Fig. 11 D two essentially planar electrodes 1 form a part of an inner surface of the chamber.
- the electrodes are es sentially opposite and thereby face each other. Between the electrodes, the electrical field is generated.
- the form of the chamber in Fig. 11 D is rectangular.
- Fig. 11 E the form of the chamber is circular, wherein in the circumferential inner housing, several electrodes 1 are located .
- Example 6 Low voltage cell lysis for DNA, RNA or protein isola ⁇ tion
- Fig. 12 A shows lysis curves of 293T cells for different conduc tivities when applying capacitive coupled electric fields with a square wave 1 kHz AC signal for 6 seconds.
- lysis begins at 10 V and reaches a plateau at 25 V.
- Increasing conductivity to 160 pS/crn we observe lysis starting below 15 V and peaking at 25 V.
- 260 pS/cm onset of lysis occurs at 20 V and reaches its maximum around 30 V.
- the dynamic range for all three conductivities spans 10 V, corresponding to 1.2 kV/cm. It was shown that lysis efficiency of 293T cells is inversely proportional to buffer conductivity. This trend is highly reproducible and lysis curves are clearly distinguished by minor changes in buffer composition.
- Fig. 12 B shows experiments with pulse number as the only variable. Field strength was set to 3.6 kV/cm and frequency at 1 kHz. As a result, lysis efficiency increases steadily with peri od number up to 2000 periods, reaching a plateau of maximum ef ficiency with around 90% lysis and minimal standard deviation for a given conductivity.
- Fig. 12 C shows 293T cell lysis upon exposure to 2000 pulses at different frequencies. While the total exposure time if sig nificantly different, adjustment to the previously determined threshold of 2000 pulses results in equal lysis efficiency inde pendent of frequency.
- Fig. 12 D shows the concentration of cell-free DNA after ly- sis of 293T cells with increasing applied voltages.
- Fig. 12 E shows the concentration of cell-free RNA after ly sis of 293T cells with increasing applied voltages.
- Fig. 12 F shows the extraction of cytosolic proteins in their native form from cells after lysis through capacitive cou pled electric fields using the pipette tip. (indorf)
- Example 7 Cargo delivery a cell transfection
- Fig. 13 A shows lysis and pro- pidium iodide (PI) delivery into 293T cells when exposed to a decreasing number of high-voltage square wave pulses. 50 pulses of 40 V lead to lysis of 48.5% of cells while PI was taken up by 78.8 % of the remaining viable cells. Decreasing the number of pulses further results in higher viability while the fraction of live cells permeable to PI remains similar. Upon exposure to 5 square wave pulses of 40 V, 5.6 % of 293T cells are lysed by the electric field while 82.8 % are viable and Pi-positive.
- PI pro- pidium iodide
- Fig. 13 B shows further optimization of Pi-delivery by changing the applied voltage.
- Cells were subjected to 5 square wave pulses at 1 kHz repetition rate. Viability is virtually un altered by the field magnitude in this pulse range. 293T cell viability is decreased by 2,5 % by application of five 25 V pulses while five 40 V pulses decrease viability by 1.8 %.
- Fig. 13 B further shows a correlation between field strength and the fraction of RI-permeable cells, yielding 22.4 %, 66.7 %, 82.3 %, 80.5 % and 81.1% for 25 V, 30 V, 35 V, 40 V and 45 V re spectively.
- Fig. 13 C shows that PI-permeabilization strongly correlates with pulse number at the same voltage. 1000 square wave pulses yield 48.4 % Pi-positive viable 293T cells, increasing up to 85.0 % at 3000 pulses with minimal loss of viability.
- Fig 13 D shows 293T cells expressing RFP 48 hours after electro transfection with pTurboRFP-N plasmid by application of exponen tial decay pulses of 40 V to the pipette tip.
- Grade 2 titanium foil (commercially pure titanium, cpTi, 99.2% pure) was cut in dimensions of 60x10 mm. An average oxide layer thickness of 500-600 nm was used. Electroporation reser voirs were assembled using double-sided 81.3 pm thick adhesive tape (Adhesive Research, Arcare 90445) spaced 2,5 mm apart form ing a 12,15 m ⁇ channel. The top 5 mm of a standard 200 m ⁇ pi pette tip was cut and a short, 90° incision was made in the re sulting narrow end.
- Electroporation tip assembly proceeded by inserting the electrode-flanked channel into the incision and sealing any edges air-tight with two-component epoxy adhesive (UHU plus Endfest 300, 45640) .
- the resulting tips can be used with any 20 m ⁇ pipette set to a volume of 10 m ⁇ .
- Human embryonic kidney cells 293T (supplied by the Depart ment of Nanobiotechnology of the University of Natural Resources and Life Sciences, Vienna) were cultivated at 37 °C and 5% C02 in DMEM (Thermo Fisher, 41965) supplemented with 10% FBS (Thermo Fisher, 10500) and 1% Pen/Strep Antibiotic -Antimycotic (Thermo Fisher, 15240) . Cells were passaged by washing with PBS (lx from stock: Thermo Fisher, 70011044) followed by trypsinization
- Electroporation buffer (EPB) was prepared from autoclaved 250 mM sucrose solution. PBS was added to adjust the desired sample conductivities. Conduc tivity was measured using a conductivity meter (B-771 LAQUAtwin, HORIBA Advanced Techno) . Cells were centrifuged for 5 min at 400 g (RT) , supernatant discarded and re-suspended with EPB. After two washing steps, cells were counted and adjusted to lxlO A 6/ml with the next reconstitution. Final conductivity was recorded.
- 293T cell lysis is used as an indirect readout to analyze the biological impact of capacitively coupled electric fields across a range of parameters.
- Cells in EP buffer were trans ferred to hydrophobic parafilm in 10 m ⁇ droplets and aspirated with the electroporation tip prototype.
- Electric fields were in prised by applying the according voltage waveforms by a function generator (DG4102, Rigol) connected to a voltage amplifier (Fal- co WMA-300, Falco Systems, Netherlands) .
- Voltages and current were monitored by an oscilloscope (DS1104B, Rigol) .
- Cells were ejected onto parafilm and mixed with a 10 g/ml (lOx) stock solution of in PBS for a final concentration of l g/ml.
- the sample was transferred to a hemocytometer (Thoma, Optik Labor) and imaged by a digital camera (Prosilica GT, Al lied Vision) mounted on an inverted microscope (CKX41 Fluo V2, Olympus) . Bright-field images were recorded for total cell count.
- Hoechst 33342 viability dye was excited at 360 nm using a UV lightsource (X-Cite 120Q, Excelitas Technologies) and emission above 420 nm imaged for further anal ysis.
- the permeabilization dye Propidium Iodide (PI) was prepared from dilution of a lmg/ml stock with electroporation buffer to limit any conductivity change.
- Cells in EP buffer were trans ferred to hydrophobic parafilm in 9 m ⁇ droplets and mixed 1:10 with a 30mm/m1 solution of PI.
- the mixture is aspirated with the electroporation tip prototype.
- Electric fields were induced by applying the according voltage waveforms by a function generator (DG4102, Rigol) connected to a voltage amplifier (Falco WMA-300, Falco Systems, Netherlands) . Voltages and current (via a 2W re sistor) were monitored by an oscilloscope (DS1104B, Rigol) .
- the sample is ejected to a hemocytometer (Thoma, Optik Labor) and imaged by a digital camera (Prosilica GT, Allied Vision) mounted on an inverted microscope (CKX41 Fluo V2, Olympus) .
- a hemocytometer Tropa, Optik Labor
- a digital camera Prosilica GT, Allied Vision
- CKX41 Fluo V2, Olympus an inverted microscope
- the membrane impermeable PI was excited from 480-550 nm using a UV lightsource (X-Cite 120Q, Excelitas Technologies) and emission above 590 nm imaged for further anal ysis.
- Lysis images were analyzed in Fiji (Schindelin et al . 2012) by adjusting the threshold to include positive cells only, iso lating high-contrast live cells in bright-field and stained dead cells in fluorescence images. After converting images to binary, cell count was performed by particle analysis function. Results are displayed as percentage lysed, excluding the fraction of dead cells from sample preparation, which means that the control is always displayed as zero percent lysis. Pi-positive cells were counted manually from a bright-field and red fluorescence overlay. Controls were overexposed to the point where PI- negative cells remain invisible. This setting is then applied to samples subjected to electric fields. Dead cells show high-PI fluorescence, look visibly dead in bright-field and are calcu lated equally as in lysis experiments. Permeabilized cells are displayed as the fraction of visibly live cells with any PI flu orescence .
- Cells prepared as in section Sample Preparation were kept in a sterile working environment. 9m1 were placed on hydrophobic parafilm and mixed with 1 m ⁇ vector stock solution for a final working concentration of 25 ng/ml pTurboRFP-N (Evrogen, FP232) and 0.5 mM MgC12. The suspension was aspirated into the electro poration tip and exposed to 10 exponential decay pulses of 40V at 1 kHz followed by 1000 exponential decay transfer pulses of 15 V at 50 Hz. They were ejected into an 8-well m-slide (Ibidi, 80826) and left to rest for 5 minutes.
- pTurboRFP-N Evrogen, FP232
- OptiMEM Thermo Fischer, 31985062
- OptiMEM 250m1 OptiMEM (Thermo Fischer, 31985062 ) was added with gentle re-suspension.
- Trans fected cells were cultivated at 37 °C and 5% C02 for 48 hours and imaged by a digital camera (Prosilica GT, Allied Vision) mounted on an inverted microscope (CKX41 Fluo V2, Olympus) .
- the fraction of cells expressing RFP was assessed from manual count of bright-field and red fluorescence overlays of at least 5 im ages from random positions in each respective well.
- Example 8 Device targeted electroporation and/or lysis of eu karyotic cellular bodies in a biological sample with at least two subgroups of eukaryotic cellular bodies for flow cytometry
- Fig. 14 shows two chambers 1 and 3 with electrodes in combi nation with a flow cytometry unit.
- the flow cytometry unit is in fluid connection with the chambers 1 and 3, so that the biologi cal sample can flow from the chambers 1, 3 to the flow cytometry unit and/or vice versa.
- a control unit 5 can be used for controlling of the flow cytometry unit and analysing the sample.
- adjustment units 2 and 4 are connected to the electrodes of the chambers.
- the control unit 5 can be connected to adjustment units 2 and 4.
- the biological sample in Fig. 14 referred to as Cell Suspension, can be mixed with a staining solution.
- Fig. 15 depicts the conductivity dependent lysis of blood cells. As shown, efficient cell lysis with capacitive coupled electric fields is efficient using a 1:10 dilution of blood in sucrose. As complete electric field induced lysis of cells hap pens in less than 6 seconds, a prototype with 1 ml volume capac ity could thus process and isolate pathogens from the required 7 ml whole blood in less than 10 minutes.
- FIG 37 a prototype design that resembles a roller-type capacitor with 1 ml sample capacity is shown.
- the complete and homoge neous coating of the electrodes by a thin film high-k material is the fundamental basis for pure physical, specific electric field effects on cells, defects in the coating result in charge transfer between the electrode and the biological sample when a voltage is applied to the electrodes, and thus, give rise to su perimposing electrochemical effects.
- the occurrence of defects in the passivation film were observed during test runs via an electrical measurement setup.
- Example 9 Application of leukocyte enrichment from human blood samples
- leukocytes were isolated from blood and exposed to dif ferent voltages and square wave frequencies in the lab-scale flow through prototype (Fig. 17) .
- a surprisingly discrete cell lysis behavior can be observed between leukocytes (magenta) and whole blood cells (red, >99% erythrocytes) dependent on the voltage amplitude (Fig. 17 a) as well as in a frequency depend ent manner (Fig. 17 b, c) .
- Leukocytes were discrimi nated from erythrocytes by using a cell permeable fluorescent dye (Hoechst) , which intercalates DNA and stains the nucleus of cells .
- Hoechst cell permeable fluorescent dye
- Example 10 Specific cancer cell ablation in mixed cell popula tions
- Liquid biopsy comprises the isolation of circulating tumor cells (CTC) from blood samples for basic cancer research and targeted treatment of cancer patients.
- CTC circulating tumor cells
- concentration of circulating cancer cells in blood is relatively low (1-10 CTCs in 10 ml blood)
- enrichment of CTCs is currently considered one of the biggest bottlenecks.
- Prior methods focus predominantly on the isolation of CTCs using anti-EPCAM antibodies.
- the strategy of using an epithelial marker on the CTCs cell surface comes with the restriction of missing cancer cells that have undergone epithelial to mesenchymal transition.
- the next application opportunity we were interested in was to transfer the strategy of using electric fields for pathogen isolation to CTC isolation from blood.
- two prominent cancer model cell lines were used.
- Jurkat cells as a model for human T cell leukemia (EPCAM negative) and MCF-7 breast cancer cells, a widely used model for EPCAM posi tive CTCs.
- FIG 20 the results of the spike-in experiment for leukemic-specific cell ablation in a mixture with healthy leuko cytes are depicted.
- Figure 20 a and b show the control flow cy tometry read out of fluorescently labeled Jurkat cells (blue) and healthy leukocytes only (magenta) , respectively.
- Figure 20 c depicts the mixed cell suspension after passing through a lab- scale flow-through prototype without an applied electric field. Clearly, both cell population can be discriminated. After appli cation of 20 V at 50 Hz sqAC, the Jurkat cell population is spe cifically depleted and an increase in cell debris (black) can be observed.
- Figure 20 e represents the relative number of flow cy tometry events from three individual experiments.
- FIG. 20 f is a representative micrograph of Jurkat cells lysed by application of 20 V 50 Hz sqAC, showing lysed cells which appear as empty cellular shells, termed "ghosts", that are grouped as debris in the flow cytometry read out.
- the Jurkat experimental design was repeated using MCF-7 breast cancer cells, a state-of-the-art CTC model cell line .
- MCF-7 cells were observed to be more susceptible to electric fields than blood cells from a healthy donor (Fig. 21) .
- the lysis rate in de pendence of voltage and frequency is also different to Jurkat cells, resulting in a smaller window of opportunity to discrimi- nate between healthy leukocytes and MCF-7 breast cancer cells using electric fields in the investigated parameter spectrum.
- MCF-7 cells showed an explicitly different lysis be havior. Instead of complete cell rupture or the appearance of empty cellular ghosts, as is observed with the cell types inves tigated during the project (erythrocytes, leukocytes, Jurkat,
- MCF-7 cells retained their intracellular granularity.
- FIG. 22 a-c are the dot plots of the flow cytometry readout for no field applied, 25 V applied at 1 kHz sqAC and 30 V applied at 100 Hz sqAC, respectively.
- Figure 22 e summarizes the results of two individual experiments. The results of the spike-in experiment show the feasibility of high ly efficient tumor cell ablation in a mixed cell suspension us ing electric fields. What can also be observed, however, is a certain degree of electric field induced perforation of healthy leukocytes with the used electric field parameters in these ex periments, as is show by the upward migration of the leukocyte population in the dot plots (Fig.
- Example 11 Application of stem cell enrichment from human blood samples
- HSC hematopoietic stem cells
- leukocyte where iso lated from blood of a healthy volunteer and exposed to electric fields through application of 40 V at 100 Hz sqAC in the lab- scale flow through prototype. Control samples were passed through the prototype without an electric field applied. Samples were then stained with fluorescently labeled antiCD45 and an- tiCD34 antibodies and analyzed in a flow cytometer.
- Figure 23 shows the result of a preliminary study on the possibility to use electric fields for the isolation of HSCs.
- 0.25% of the total cell suspension in the control sample are events that are CD45 and CD34 positive and thus represent HSCs. This value is in the expected range for the frequency of HSCs in blood.
- the number of CD45 and CD34 positive events is in creased approximately 10 fold to 2.2% of the total events.
- the results suggest that an enrichment of HSC from blood could be feasible via the exploitation of different susceptibilities of hematopoietic stem cells and leukocytes. It has to be indi cated, however, that these results will include different sets of antiCD45 antibodies.
- Example 12 Electrode design for gene transfer
- electroporation Besides cell lysis through electric fields, termed irre versible electroporation, another highly interesting effect of electric fields is the generation of temporary pores in the cell membrane induced via an electric field, termed reversible elec troporation. Such temporary pores are widely used to introduce foreign molecules into cells, majorly DNA plasmids for genetic manipulation of cells, i.e. transfection of eukaryotic cells. Although viral, chemical and physical transfection technologies are available, electroporation potentially offers unique ad- vantages, such as low cost, high throughput, specificity and controllability. But still, the major challenges facing current electroporation products are technological and operational com plexity, system scalability, cell viability and reproducibility.
- the equivalent circuit was transferred to an electri cal circuit design and simulation program.
- Several electric pa rameters were feed into the equivalent circuit to observe tem poral potential drops over individual circuit elements such as the electrolyte.
- electric settings from performed cell lysis experiments were compared to add detailed electrical parameters to a profound data set of over 700 indi vidual HER cell lysis data points.
- the model could plausibly pre dict lysis rates if different conductive solutions are given.
- four independently performed lysis experiments at dif ferent voltages, frequencies and exposure times were performed and compared with the prediction of the model.
- Example 15 Chamber with electrodes with Semiconductor charac teristics .
- Thermally generated titan oxide was investigated for is in sulating or n-type semiconductor behaviour.
- a I/V scan was per formed for non-passivated and passivated electrodes. Therefore, electrodes were contacted via the base metal and via a silver contact paste covering a surface area of 90 mm 2 . Current was measured at corresponding DC voltages applied.
- titanium electrodes without thermal oxidation show a linear increase in current with the ap plied DC voltage independent of polarity, thus representing a resistive element.
- passivated titanium electrodes show a current blocking behavior when applying negative poten tials, thus acting with n-type semiconducting characteristics.
- a titanium electrode with deficient passivation layer shows die lectric breakdown when voltages above 12 V are applied. As such, the Ti02 layer on top of the electrode represent a diode, block ing the current in one direction.
- one passivation layer acts as high resistive element and the other as low resistive element, which is reversed when the electric potential is reversed, only the high resistive ele ment experiences a significant potential drop. This allows for a larger proportion of the potential to drop in the fluid, thus a higher electric field, but still providing capacitive decoupling of the electrode material with the liquid sample.
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